Nitric oxide (NO), which can be produced intracellularly by endogenous NO synthase (NOS), acts as an important second messenger. The known functions of NO include regulation of smooth muscle relaxation, platelet aggregation, neurotransmission, neurotoxicity, cell death and differentiation. In order to promote smooth muscle relaxation in vasculature, NO targets the heme group located at the active-site of soluble guanylyl cyclase, resulting in elevation of its enzymatic activity, consequently leading to vasorelaxation.
For executing other biological functions, NO is likely to react with cysteine (Cys) residues in proteins. The reaction of NO with cysteine residues in proteins to form S-nitrosothiol (SNO) is a post-translational modification process known as protein nitrosation or S-nitrosylation. Protein S-nitrosylation regulates the activity of a large number of targets, including metabolic, structural, cytoskeletal, and signaling proteins, and is an important mechanism for nitric oxide signaling.
Although significant progress has been made to understand the biological function of protein S-nitrosylation, the role of nitrosylation in regulating signal transduction has not been well characterized. The detection of protein S-nitrosylation in complex biological system remains challenging due to technical limitations of current methods, and the unstable nature of S—N bonds in S-nitrosothiols (SNO).
Current methods known in the arts for the detection of SNO include indirect detection methods and direct detection methods. An immunohistochemical approach using anti-S-nitrosocysteine antibody is a direct method for detection of protein S-nitrosylation. Yet, as the unstable S—N bonds may be broken during SDS-PAGE separation in immunoprecipitation or Western blotting, the method is not typically performed. Indirect methods usually break the unstable S—N bonds and capture either the sulfur or the nitrogen part for detection. Among methods for studying protein S-nitrosylation, the biotin switch method (Jeffery (2001) Nat Cell Biol, 3: 193-197) has become a mainstay assay due to the ease with which it can detect individual S-nitrosylated (SNO) proteins in biological samples. Expression of endogenous biotin in various tissues including kidney, liver and brain has been well documented in literatures (Wang (1999) Cell Tissue Res, 296: 511-516; McKay (2004) J Comparative Neurology, 473: 86-96). It was also shown that mitochondrial matrix contains a significant level of biotinylated proteins (Hollinshead (1997) J Histochem Cytochem, 45: 1053-1057). The presence of endogenous biotin and biotinylated proteins may cause unexpected background signals in any application of biotin-avidin or biotin-streptavidin technique, including the biotin-switch method. The problems caused by endogenous biotin and biotinylated proteins suggest that many false-positive results have been generated by the biotin-switch method since its introduction 15 years ago.
Insulin delivery to the skeletal muscle interstitium plays a key role in insulin-directed glucose uptake by skeletal muscle. This insulin signaling-dependent process is tightly controlled by the endothelial barrier function of the capillaries connected to skeletal muscle. It has been shown that intrinsic NO functions to promote insulin delivery across endothelial barrier. However, to date the mechanism underlying NO-dependent insulin responsiveness in endothelium remains uncharacterized due to technical limitations of current detection methods for protein S-nitrosylation.
Thus, there remains an unmet need in the art for a new analytic platform to enhance the specificity and accuracy of the detection of S-nitrosylated proteins and facilitate the study of S-nitrosylation's role in signal transduction pathways.